Background: Most clinical guidelines for the use of biocides have been developed for planktonic micro-organisms, but in nature, most micro-organisms live as surface-adherent communities or biofilms.

Aim: To evaluate the effectiveness of commonly used biocides against . Escherichia coli and . Candida spp. in three distinct growth phases: planktonic, adhesion and biofilm.

Methods: Ultrastructural, architectural and cellular viability changes following a 5 min exposure to biocide were monitored by scanning electron microscopy and confocal laser scanning microscopy using fluorescent dyes. Comparative transcript expression of the antioxidants . SOD1 and . CAT1 in the planktonic and biofilm phases was evaluated using quantitative real-time polymerase chain reaction.

Findings: E. coli and . Candida spp. in the planktonic phase were susceptible to all the tested biocides at the recommended concentrations. However, early adhesion and late biofilm phases of both were less susceptible to the biocides, and exceeded the recommended concentrations on several occasions. A short period of biocide exposure failed to fully eradicate the adherent microbial cells, and they recovered from the biocide challenge, forming biofilm on the biocide-treated surfaces. The biofilm phase showed higher expression of . SOD1 and . CAT1.

Conclusion: The recommended concentrations of biocides for clinical disinfection in the hospital setting may not fully eradicate the adhesion or biofilm phases of . E. coli and . Candida spp. Higher antioxidative capacities in microbial biofilms may be responsible for the resistance of biofilms against clinical biocides.